Method for suppressing growth of mycobacteria in metalworking fluids

ABSTRACT

A method for suppressing the growth of mycobacteria associated with hypersensitivity pneumonitis, in particular  Mycobacterium immunogenum , in metalworking fluids is described. The method uses antimicrobial or biocidal compositions which include a metal complex comprising disodium monocopper (II) citrate and an isothiazolone mixture preferably comprising 5-chloro-2-methyl-4-isothiazolin-3-one 2-methyl-4-isothiazolin-3-one. Also described are methods for detecting and identifying the mycobacteria.

CROSS-REFERENCE TO RELATED APPLICATION

Not applicable.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

REFERENCE TO A “COMPUTER LISTING APPENDIX SUBMITTED ON A COMPACT DISK”

Not Applicable.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to a method for suppressing the growth ofmycobacteria associated with hypersensitivity pneumonitis, in particularMycobacterium immunogenum, in metalworking fluids. The method usessynergistic antimicrobial or biocidal compositions which include a metalcomplex comprising disodium monocopper (II) citrate and an isothiazolonemixture preferably comprising 5-chloro-2-methyl-4-isothiazolin-3-one2-methyl-4-isothiazolin-3-one. The present invention also relates tomethods for detecting and identifying the mycobacteria.

(2) Description of Related Art

The prior art has described metal complexes of organic ligands asantimicrobial or biocidal compounds. These include U.S. Pat. No.4,055,655 to Maurer et al., U.S. Pat. No. 4,129,589 to Shringarpurey etal., and U.S. Pat. No. 4,180,473 to Maurer et al. The process for theirmanufacture is described in U.S. Pat. No. 4,278,610 to Maurer et al. Theproblem is that these compounds are relatively poor antimicrobials andeven large amounts provided protection for only a limited period oftime.

The isothiazolones are described in U.S. Pat. No. 3,523,121 to Lewis etal.; U.S. Pat. No. 3,761,488 to Lewis et al.; U.S. Pat. No. 3,957,808 toMiller et al.; U.S. Pat. No. 4,105,431 to Lewis et al.; U.S. Pat. No.4,243,403 to Lewis et al.; U.S. Pat. No. 4,252,694 to Lewis et al.; U.S.Pat. No. 4,265,899 to Lewis et al.; U.S. Pat. No. 4,279,762 to Lewis etal. These are very superior antimicrobial agents; however, relativelylarge amounts are required. Compatibility and stabilization ofisothiazolones in metalworking fluids (MWF) has been described byWillingham and Derbyshire, J. Soc. Tribol. Lub. Eng. 47: 729-732 (1991).

Disodium monocopper (II) citrate (MCC) is particularly described as anantimicrobial compound by U.S. Pat. No. 4,055,655. Metalworking fluid(MWF) stabilizing activity is described in U.S. Pat. No. 4,129,509(1978)). The former patent states that the compound is effective againstmicroorganisms growing in alkaline environments (pH 8-12) due to thestability of the metal complex form only at high pH, with dissociationinto toxic copper ions occurring upon encountering the lower pH (7.0)within microbial cells.

Studies on MCC have shown that it can temporarily inhibit the growth ofPseudomonas aeruginosa in laboratory media and transiently reduce thecell count in MWF contaminated with Pseudomonas spp. The use of MCC as aMWF additive is becoming more widespread and an improvement in itseffectiveness was needed.

Although bacteria are highly important in the biodeterioration of MWF,fungi and yeast can play a major role as well, especially in thesynthetic fluids (Bennett, Prog. Indust. Microbial., 13: 121 (1974)),(Rossmoore and Holtzman, Dev. Indust. Microbiol., 15: 273-280 (1974)).Fusarium and Cephalosporium are prominent fungal contaminants, and amongthe yeasts, Candida and Trichosporon spp. are often isolated. Fungi andyeast are known to be sensitive to the toxic effects of Cu ion (Hugo andRussell, In: Principles and Practices of Disinfection, Preservation andSterilization, Russell et al. (Eds.), Blackwell Scientific Publications,Boston, p. 69 (1982)) and consequently the effect of MCC at high pH on arepresentative yeast, Candida tropicalis, was studied.

As a result of the machining operation itself, MWF can becomecontaminated with selectively large concentrations of soluble iron. Thehigh stability constant of ferric citrate can allow exchange reactionsbetween the ferric and copper ions in binding to the citrate ligand(Ashcroft and Mortimer, Thermochemistry of Transition Metal Complexes,Academic Press, New York (1970)). Such reactions may destroy theantimicrobial activity of MCC in alkaline environments.

KATHON 886 is a commercial antimicrobial solution which is a mixture of5-chloro-2-methyl-4-isothiazolin-3-one (8.6% by wt.) and2-methyl-4-isothiazolin-3-one (2.6% by wt.). The mixture is veryeffective against bacteria, fungi and algae. The required dosages aresometimes ineffective in achieving the best results due to interferingnucleophiles in the metalworking fluids. The molecular speciesconsidered as nucleophiles in these systems are amines and sulfides, theformer contributed by many metalworking fluid formulae and the latterfrom microbial activity. Thus, efficacy is a function of themetalworking fluid composition as well as the level of microbialcontamination, nucleophiles from both competing for the isothiazolones.

An important improvement in methods for suppressing growth ofmicroorganisms in MWFs was introduced by U.S. Pat. No. 4,608,183 toRossmoore. The patent discloses novel antimicrobial or biocidal mixturescomprising disodium monocopper citrate (MCC) and a mixture ofisothiazolones. The components in the mixture were found tosynergistically suppress growth of many species of bacteria and fungi inMWF over a long period of time. The synergism has been described inRossmoore, Intl. Biodeter. 26: 225-235 (1990) and Sondossi et al., Intl.Biodeter. Biodegr. 32: 243-261 (1993). The compositions are particularlyuseful for metal cutting fluids wherein long duration antimicrobialactivity is desired.

Over the past decade, it has been recognized that MWFs can becontaminated with mycobacteria as well as bacteria and fungi. It wasfurther recognized that high levels of mycobacteria in MWFs appeared tocoincide with outbreaks of hypersensitivity pneumonitis (HP) inindustrial machinists (Kreiss and Cox-Ganser, Am. J. Ind. Med. 32:423-432 (1997); Shelton et al., Emerg. Infect. Dis. 5: 270-273 (1999);Moore et al., AIHJ 62: 205-213 (2000)). Recently, a newly describedspecies of mycobacteria, Mycobacobacterium immunogenum, was identifiedin MWFs associated with outbreaks of HP (Wilson et al., Int. J. Syst.Evol. Microbiol. 51: 1751-1764 (2001). More recently, a single genotypeof Mycobacterium immunogenum was discovered to be present in MWFsassociated with HP (Wallace et al., Appl. Environ. Microbiol. 68:5580-5584 (2002)). A sample of the Mycobacterium immunogenum isolatedfrom MWF was deposited at the American Type Culture Collection, 10801University Boulevard, Manassas, Va. as ATCC 700506 and the DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig,Germany as DSMZ 8223. Outbreaks of HP has continued even though MWFs areroutinely treated with biocides. Therefore, there is a need for a methodfor treating MWFs which will suppress growth in MWFs the Mycobacteriumimmunogenum genotype responsible for the HP outbreaks. Preferably, themethod would suppress growth of other bacteria and fungi as well.

SUMMARY OF THE INVENTION

The present invention provides a method for suppressing the growth ofmycobacteria associated with hypersensitivity pneumonitis, in particularMycobacterium immunogenum, in metalworking fluids. The method usessynergistic antimicrobial or biocidal compositions which include a metalcomplex comprising disodium monocopper (II) citrate and an isothiazolonemixture preferably comprising 5-chloro-2-methyl-4-isothiazolin-3-one2-methyl-4-isothiazolin-3-one. The present invention also provides amethod for-detecting and identifying the mycobacteria.

Therefore, the present invention provides a method for suppressinggrowth of mycobacteria associated with hypersensitivity pneumonitis inan industrial metalworking fluid which comprises (a) providing acomposition comprising a copper complex comprising disodium monocopper(II) citrate and an isothiazolone mixture comprising5-chloro-2-methyl-4-isothiazolin-3-one and2-methyl-4-isothiazolin-3-one, wherein the copper complex providescopper ion to the metalworking fluid in an amount which maintainsbiocidal activity of the isothiazolone mixture; (b) adding thecomposition to the metalworking fluid in an amount which is sufficientto suppress the growth of the mycobacteria in the metalworking fluid forat least 48 hours; and (c) monitoring the metalworking fluid containingthe composition for the mycobacteria.

The present invention further provides a method for suppressing growthof mycobacteria in a metalworking fluid which comprises (a) providing acopper complex comprising disodium monocopper (II) citrate and anisothiazolone mixture comprising 5-chloro-2-methyl-4-isothiazolin-3-one2-methyl-4-isothiazolin-3-one, wherein the copper complex provides morethan 250 ppm, preferably about 500 ppm, of copper ion to themetalworking fluid; (b) adding the copper complex and the isothiazolonemixture to the metalworking fluid wherein the metal complex and theisothiazolone mixture suppresses the growth of the mycobacteria in themetalworking fluid for at least 48 hours; and (c) monitoring themetalworking fluid containing the metal complex and isothiazolone forthe mycobacteria.

In a further embodiment of the above methods, the copper complex isadded to the metalworking fluid up to about 24 hours before theisothiazolone mixture is added to the metalworking fluid or the coppercomplex and the isothiazolone mixture are added to the metalworkingfluid at the same time.

In a preferred embodiment of the above methods, the5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-oneare in a weight ratio of about 3 to 1. It is further preferable that thecomposition be mixed with the metalworking fluid to produce an amount ofthe isothiazolone mixture which is greater than about 7.5 ppm,preferably from about 15 ppm to about 21 ppm. The above methods areparticularly efficacious against the mycobacteria Mycobacteriumimmunogenum which has a restriction fragment length polymorphism patternproduced by a DraI digest of Mycobacterium immunogenum DNA as shown inFIG. 1. In further embodiments, the copper complex provides more than250 ppm, preferably about 500 ppm, copper ion to the metalworking fluid.The composition is efficacious even when introduced to MWFs in which theisothiazolone is incompatible with the metalworking fluid when theisothiazolone mixture is introduced into the metalworking fluid alone.

In a further embodiment of the above methods, the isothiazolone mixturecomprises 5-chloro-2-methyl-4-isothiazolin-3-one2-methyl-4-isothiazolin-3-one, wherein the isothiazolone mixture ispresent in an amount which is less than is required for biocidalactivity when the mixture is introduced into the metalworking fluidalone. Preferably, the copper complex provides more than 250 ppm,preferably about 500 ppm, of copper ion to the metalworking fluid.

The present invention further provides a method for reducing a risk thata person in a metalworking environment will contract hypersensitivitypneumonitis caused by mycobacteria which is present in the environment,which comprises (a) providing a composition comprising a copper complexcomprising disodium monocopper (II) citrate and an isothiazolonemixture; (b) adding the composition to metalworking fluids in theenvironment in an amount which is sufficient to suppress the growth ofthe mycobacteria in the metalworking fluid for at least 48 hours whereinthe metalworking fluid containing the composition is monitored for themycobacteria; and (c) exposing the person to the metalworkingenvironment, wherein suppression of the growth of the micobacteria inthe metalworking fluid by the composition reduces the risk that theperson will contract the hypersensitivity pneumonitis.

The present invention further provides a method of industrial metalgrinding for producing a shaped metal article which provides a reducedrisk of hypersensitivity pneumonitis in a metal-grinding machinist,which comprises (a) applying a force to a metal workpiece with agrinding tool contacting the workpiece while supplying to the interfacebetween the grinding tool and the workpiece a water-based metalworkingfluid comprising a copper complex comprising disodium monocopper (II)citrate and an isothiazolone mixture wherein the metal complex and theisothiazolone mixture is in an amount which is sufficient to suppressgrowth of mycobacteria associated with the hypersensitivity pneumonitisin the metalworking fluid for at least 48 hours; and (b) producing theshaped metal article from the metal workpiece while supplying themetalworking fluid to the interface wherein the copper complex andisothiazolone mixture in the metalworking fluid enables themetal-grinding machinist to produce the shaped metal article with thereduced risk of the hypersensitivity pneumonitis.

In a further embodiment of the above methods, the isothiazolone mixturecomprises 5-chloro-2-methyl-4-isothiazolin-3-one2-methyl-4-isothiazolin-3-one, wherein the mixture is present in anamount which is less than is required for biocidal activity when themixture is introduced into the metalworking fluid alone and wherein thecopper complex provides more than 250 ppm, preferably about 500 ppm, ofcopper ion to the metalworking fluid.

In a preferred embodiment of any one of the above methods, the5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-oneare in a weight ratio of about 3 to 1. It is further preferable that thecomposition be mixed with the metalworking fluid to produce an amount ofthe isothiazolone mixture which is greater than about 7.5 ppm,preferably from about 15 ppm to about 21 ppm. The above methods areparticularly efficacious against the mycobacteria Mycobacteriumimmunogenum which has a restriction fragment length polymorphism patternproduced by a DraI digest of Mycobacterium immunogenum DNA as shown inFIG. 1. In further embodiments of any one of the above methods, thecopper complex provides about more than 250 ppm, preferably about 500ppm, copper ion to the metalworking fluid. The composition used in anyone of the above methods is efficacious even when introduced to MWFs inwhich the isothiazolone is incompatible with the metalworking fluid whenthe isothiazolone mixture is introduced into the metalworking fluidalone.

The present invention further provides a method for determining thepresence or absence of an industrial metalworking fluid isolate ofMycobacteria immunogenum in a sample, which comprises (a) culturing thesample in a medium which supports growth of the isolate; (b) isolatinggenomic DNA from the isolate which grows in the culture; (c) digestingthe genomic DNA with DraI restriction enzyme to produce largerestriction fragments of the genomic DNA; (d) electrophoresising thelarge restriction fragments on a gel by pulse-field gel electrophoresisto produce a pattern of the large restriction fragments on the gel; and(e) analyzing the pattern of the large restriction fragments wherein apattern as shown in FIG. 1 indicates the presence of the isolate in thesample. In a further embodiment, the medium comprises a biocide whichcomprises a mixture of isothiazolones.

The present invention further provides a method for determining thepresence or absence of an industrial metalworking fluid isolate ofMycobacteria immunogenum in a sample, which comprises culturing thesample in a medium comprising a tryptic soy or Middlebrook 7H10 medium,wherein growth of the isolate in the medium in less than seven days atabout 30° or 35° C. indicates the presence of the isolate in theindustrial metalworking fluid. Preferably, the medium further includesTween 80, glycerin, cyclohexamide, chloramphenicol, and gentimicin. Itis particularly convenient to provide the assay as a kit wherein an agarcontaining the medium is provided as the substrate for growing themycobacteria. The agar containing the medium can be provided on petridishes or the like or in a vial as an agar slant.

OBJECTS

The object of the present invention is to provide a method which reducesthe risk that a metalworking machinist will contract hypersensitivitypneumonitis a metalworking environment.

A further object of the present invention is to provide a method ofindustrial metal grinding for producing shaped metal articles whereinthe risk of a metal-grinding machinist contracting hypersensitivitypneumonitis is reduced.

A particular object of the present invention is to provide a method inwhich growth of Mycobacterium immunogenum in metalworking fluids issuppressed.

These and other objects of the present invention will becomeincreasingly apparent with reference to the following drawings andpreferred embodiments.

DESCRIPTION OF THE DRAWING

FIG. 1 shows a pulse-field gel electrophoresis (PFGE) pattern ofMycobacteria immunogenum from MWF from different metalworking plants.The chromosomal DNA was digested with DraI. Lanes: 1 yeast DNAstandards; isolates from MWF in a plant in: 2, Ohio; 3, Illinois; 4,Indiana; 5, Wisconsin; 6, Illinois; 7, Wisconsin; 8, Indiana; 9,Michigan; and, 10, New York.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, government publications, governmentregulations, and literature references cited in this specification arehereby incorporated herein by reference in their entirety. In case ofconflict, the present description, including definitions, will control.

U.S. Pat. No. 4,608,183 to Rossmoore discloses novel antimicrobial orbiocidal mixtures comprising disodium monocopper citrate (MCC) and oneor more isothiazolones which in the mixture synergistically suppressgrowth of many species of bacteria and fungi in metalworking fluids(MWF) over a long period of time. The mixtures were found to beparticularly useful for treating MWFs wherein it was desired to conferlong duration antimicrobial activity to the MWF even when theisothiazolones were at a concentration which if provided to the MWFwithout the MCC would not have been biocidal. Rossmoore (Intl. Biodeter.26: 225-235 (1990) and Sondossi et al. (Intl. Biodeter. Biodeg. 32:243-261 (1993) describe the interaction of isothiazolones and copper andthe factors involved in the biocidal activities of isothiazolonemixtures. However, in spite of the widespread use of these mixtures tosuppress microbial growth in MWFs, a recently characterized disease,hypersensitivity pneumonia, has emerged and is now a problem in themachine industry. More recently, a particular genotype of Mycobacteriumimmunogenum (MWF genotype) has been determined to be the etiologicalagent for hypersensitivity pneumonia (Wallace, Jr. et al., Appl.Environ. Microbiol. 68: 5580-5584 (2002).

I have discovered that contamination of MWF with the MWF genotype ofMycobacterium immunogenum can be controlled when a copper metal complexsuch as the preferred disodium monocopper citrate (MCC) is provided tothe MWF in a mixture which comprises one or more isothiazolones or isprovided to the MWF prior to the addition of the isothiazolones. Themixture suppresses the growth of the MWF genotype in the MWF.Suppression of mycobacteria growth is particularly efficacious when thecopper metal complex is provided at a concentration greater than 250ppm, preferably, at a concentration of about 500 ppm or more and theisothoazolone is provided at a concentration greater than 7.5 ppm,preferably at a concentration of about 15 ppm or more. In addition, MWFscontaining the copper metal complex in an amount greater than 250 ppm,for example 500 ppm, stabilizes the isothiazolones for at least 48hours.

As used herein, the term “suppression” when referring to mycobacteriagrowth in MWF means any one of the following activities: limiting thegrowth of the mycobacteria, inhibiting the growth of the mycobacteria,stopping the growth of the mycobacteria, inactivating the mycobacteria,or killing the mycobacteria.

The time in which the copper metal complex is added to the MWF relativeto the time the isothiazolones are added to the MWF determines whethergrowth of the Mycobacterium immunogenum MWF genotype is suppressed. Itwas observed that adding MCC to a MWF 24 hours after the isothiazoloneshad been added to the MWF was ineffective at suppressing growth of theMycobacterium immunogenum genotype. Thus, for proper supression ofmycobacteria growth in the MWF, the copper metal complex should be addedto a MWF within 24 hours of addition of the isothiazolones. However, toensure effective suppression of mycobacteria growth, it is preferablethat the copper metal complex be added to the MWF prior to the additionof the isothiazolones. Most preferably, the copper metal complex isadded to the MWF at the same time the isothiazolones are added to theMWF. Therefore, in the most preferred embodiment, a mixture is providedas a single product which contains both the copper metal complex and theisothiazolones. The product provides a simple and convenient means fortreating MWFs to suppress growth of the mycobacteria. A productcomprising a mixture of both the copper metal complex and theisothiazolone is preferred because it prevents the occurrence ofsituations in which the isothiazolones are added to the MWF before thecopper metal complex is added to be avoided. In other words, the mixtureof copper metal complex and isothiazones ensures that the MWF containsboth the copper metal complex and the isothiazones and that theisothiazolones were not added to the MWF prior to the copper metalcomplex.

The above mixture was further discovered to be efficacious even in MWFswhich are incompatible with isothiazolones. That is, the MWF containsone or more components or variables which interfere with theantimicrobial activity of the isothiazolones, either partially orsubstantially reducing the efficacy of the isothiazolones to suppressgrowth of microorganisms (bacteria, fungi, and mycobacteria). In someinstances this is interference is effected by various components in theMWF which facilitate conversion of isothiazolones from a chlorinatedform to an unchlorinated form. Incompatibility of a MWF forisothiazolones can be effected by such variables as high pH-values(greater than about 8.5) or low pH-values (less than about 5.0); hightemperatures (greater than about 40° C.); various reducing and oxidizingsubstances such as hypochloride, bisulfite or H₂S, cysteine, and thelike; and, various strong nucleophilic substances (depending on pH andtemperature) such as ammonia, primary and secondary amines, sulfides,and the like. Thus, the above mixture can be used to suppress the growthof the Mycobacterium immunogenum isolate even in MWFs which areincompatible with biocides comprising isothiazolones.

As shown in Examples 1-4, the copper metal complex must be added to theMWF in the appropriate amount and before adding the isothiazolones inthe appropriate amount to the MWF. This is necessary in order for theisothiazolones to be stablized in the presence of nucleophiles which maybe present in the MWF and which in turn maintains the efficacy of thecomposition against the mycobacteria. However, there has been apractical difficulty in guaranteeing that the copper metal complex willbe present in industrial MWF systems when needed and at the appropriateamount. This is because even though written protocols have been providedwith the metal copper complex and isothiazolones which explain the needfor adding the copper metal complex to the MWF before the isothiazolonesare added to the MWF, the copper metal complex is often not added to theMWF until after the isothiazolones have been added to the MWF and thatis even when it is added to the MWF before there is both physical andchemical depletion of the copper in the MWF preventing its appropriateaction. Therefore, in a preferred embodiment of the present invention, acomposition is provided which comprises a mixture of the copper metalcomplex and the isothiazolones, each in an amount which enables thecomposition when added to a MWF to suppress growth of mycobacteria inthe MWF, in particular the MWF isolate of Mycobacterium immunogenum.

The preferred composition avoids the above practical difficulty whereinthe isothiazolones are added to a MWF before the copper metal complex isadded to a MWF. The preferred composition can be provided in a singlecontainer which contains both the copper metal complex and theisothiazolones, each in an amount appropriate for treating a particularvolume of MWF. In other words, the amount of composition in eachcontainer is sufficient to treat a particular volume of MWF. Inpractice, the user measures out from the container the appropriateamount of composition to be added to a particular volume of MWF tosuppress growth of mycobacteria in the particular volume of MWF. Forparticularly large volumes of MWF, several containers of the compositionmight be added to the MWF to provide the proper amount of composition tosuppress growth of the mycobacteria in the MWF. Preferably, thecomposition is provided in a concentrated form such as 2×, 5×, 10×, orthe like. The concentrated form facilitates storage and handling of thecomposition. In another embodiment, the copper metal complex andisothiazolones are provided in separate containers with instructionsthat the copper metal complex and isothiazolones are mixed before addingto the MWF. This can be accomplished by the user measuring out theproper amounts of each for a particular volume of MWF, mixing themtogether in a vessel, and then adding the mixture in the vessel to theMWF. A mixing vessel can be included with the separately provided coppermetal complex and isothiazolones.

The preferred copper metal complex is disodium monocopper (II) citrate(MCC) which is available as a concentrated commercial preparation fromCoolant Control, Inc., Cincinnati, Ohio. Analysis via the iodidereduction method revealed a concentration of 1 mole/liter of Cu ion. Thepreparation was sterilized by passage through an 0.22 μm membranefilter. Copper containing compounds from Coolant Control, Inc. which aresold as enhancers for isothiazolone-based biocides include CUPRISAN,CITROXYL, and OXCEDOT 0-50 (disodium monocopper (II) citrate).

Isothiazolones are frequently provided as a mixture of isothiazolones.For example, chloromethylisothiazolone (CMIT) is a mixture of5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin whichis available under the trademark KATHON from Rohm and Haas ofPhiladelphia, Pa. KATHON includes KATHON 893 and KATHON 886 which arealso available from Coolant Controls, Inc. In virgin KATHON, the rationof chlorinated to unchlorinated isothiazolones is about 3 to 1. Thechlorinated form is the most active component of the isothiazolonemixture and it is also the most nucleophile sensitive. In many MWFs theratio shifts over time towards the unchlorinated form because ofreduction by one or more components which might be present in the MWF.The MCC appears to inhibit the dechlorination of the KATHON.

Mycobacterium immunogenum isolated from a MWF has been deposited at theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. as ATCC 700506 and the Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH, Braunschweig, Germany as DSMZ 8223. The phenotypiccharacteristics of the isolate is described in (Moore et al., AIHJ 61:205-213 (2000); Wilson et al., Intl. J. Syst. Evol. Microbiol. 51:1751-1764 (2001)). Briefly, the Mycobacterium immunogenum isolate has atypical morphology with acid-fast staining, the absence of pigmentation,growth on tryptic soy and Middlebrook 7H10 agar in less than seven daysat 30° and 35° C., no growth at 45° C., mycolic acid production, growthon MacConkey agar without crystal violet, negative for iron up-take andnitrate reductase, and a positive 3-d arylsulfatase reaction. Theisolate cannot utilize citrate, D-glucitol (D-sorbitol), i-myo-inositol,and D-mannitol as sole carbon sources. The isolate is resistant toamikacin (MIC 128 μg/mL), cefmetazole (MIC 64 μg/mL), cefoxitin (MIC 64μg/mL), ciprofloxacin (MIC >16 μg/mL), doxyxycline (MIC >128 μg/mL),imipenem (MIC 16 μg/mL), sulfamethoxazole (MIC >64 μg/mL), andtobramycin (MIC >16 μg/mL). The isolate is unable to grow onLöwenstein-Jensen medium containing 5% NaCl at 35° C. However, theisolate exhibits 25 to 50% growth in the presence of 5% NaCl at 30° C.compared with control growth on Löwenstein-Jensen medium containing 5%NaCl at 35° C. General methods for identifying mycobacteria includeTelenti et al., J. Clin. Microbiol. 31: 175-178 (1993); Wallace Jr. etal., J. Clin. Microbiol. 31: 2697-2701 (1993); Wallace Jr. et al., J.Clin. Micobiol. 31: 3231-3239 (1993); and, Steingrube et al., J. Clin.Microbiol. 33: 149-153 (1995). The methods described in the above havebeen used to characterize the MWF isolate disclosed herein.

Mycobacterium immunogenum isolated from MWFs has further been describedin Wallace Jr. et al., Appl. Environ. Microbiol. 68: 5580-5584 (2002)which discloses that Mycobacterium immunogenum isolated from MWFs from aplurality of locations across the United States all have a particulargenotype. These MWF isolates were found to have an identical DraIrestriction enzyme pattern when their genomic DNA was digested with DraIand resolved by pulse-field gel electrophoresis (See FIG. 1). This DraIrestriction enzyme pattern distinguishes the MWF isolates fromMycobacterium immunogenum isolated from other sources.

The above characteristics of the MWF isolate of Mycobacteriumimmunogenum can be used to design a diagnostic assay for determining thepresence of the isolate in a MWF. For example, a sample from a MWF isincubated in a tryptic soy or Middlebrook 7H10 medium which preferablyfurther includes Tween 80, glycerin, cyclohexamide, chloramphenicol, andgentimicin to suppress growth of unwanted bacteria and fungi. Growth ofthe isolate in the medium indicates the presence of the isolate in theindustrial metalworking fluid. Particularly, when multiple samples areassayed and there is growth in less than seven days for the samplesincubated at about 30° and 35° C. and no growth for the samplesincubated at about 45° C. The assay can further include positive andnegative controls. The assay can be performed in liquid medium, however,it is preferable to perform the assay on an agar support which comprisesthe medium. For example, a sample of MWF is inoculated onto agar platesor slants which comprise the medium. The inoculated plates or slants arecovered and incubated at the appropriate temperature. Detection of themycoplasma is by visually observing by eye colonies of the mycobacteriawhich can have either a rough or smooth morphology. Alternatively, themycobacteria can be visualized by acid-fast staining.

The above assay can be provided as a kit. In one embodiment, a kit isprovided in which agar comprising the above medium is provided as anagar slant in a container which has a lid (screw cap or snap-top). Thisembodiment of the kit enables the user to inoculate the agar slant inthe container, cover the container with the lid, and incubate thecontainer at the appropriate temperature with monitoring for growth ofthe mycobacteria. In further embodiments, the kit can include a sampleapplicator. In a second embodiment, a kit is provided in which flexibleplastic strips to which absorbant material such as filter papercomprising the above medium is attached. A MWF sample is inoculated tothe filter paper on the strip or the filter paper on the strip isinoculated by dipping into the MWF. Afterwards, the inoculated strip isincubated at the appropriate temperature and monitored for growth of themycobacteria. In a third embodiment, a kit is provided in which a slideis afixed to the inner surface of a lid to a container with an opening.When the lid is fastened to the container, the slide extends from thelid into the opening of the container. The slide comprises on itssurface the above medium in agar or absorbant material. A MWF sample isinoculated to the slide or the slide is inoculated by dipping into theMWF. The slide is then inserted into the opening of the container andthe lid fastened. The container with the slide therein is incubated atthe appropriate temperature with monitoring for growth of themycobacteria. The above kits can be provided with the above productcomprising the mixture of the copper metal complex, preferably the MCC,and isothiazolones, preferably the mixture of5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin.

The following examples are intended to promote a further understandingof the present invention.

EXAMPLES 1 AND 2

This example illustrates that in order for the mixture to be efficaciousagainst Mycobacterium immunogenum MWF isolates, the copper activity fromthe copper metal complex needs to be present in industrial MWF systemseither prior to addition of the isothiazolones or the copper metalcomplex is mixed with the isothiazolones and the mixture is added to theMWF. When the isothiazolones are added 24 hours before the copper metalcomplex is added, there is no protection. In spite of instructions toon-site workforces about the order and time of addition of the coppermetal complex and the isothiazolones, frequently the isothiazolones areadded before the copper metal complex. The result is no biocidalprotection against mycobacteria.

The MWFs selected were fluids which had evidence of CMITincompatibility. The MCC was added to the MWFs 24 hours before the CMITwas added; 24 hours after the CMIT was added; or in a mixture with theCMIT. This was done for each concentration of CMIT (7.5 and 15 ppm) andMCC (250 and 500 ppm). Controls consisted of no biocide and CMIT at 7.5and 15 ppm. Stability of the isothiazolones was evaluated at time 0 andthen after 24 and 48 hours by standard methods for detectingisothiazones. Plating for mycobacteria kill was performed with aliquotsfrom time 0 and 48 hours.

Plating was performed on 100 mm petri dishes containing 20 mL of anappropriate agar. Two different formulations, both based on Plate CountAgar (standard methods for waste and waste water). The medium forrecovery of Mycobacterium immunogenum is nutritionally supplemented withTWEEN 80 and glycerin and has three antibiotics (cyclohexamide,chloramphenicol, and gentimicin) to inhibit fungi and unwanted bacteria.This medium with agar is referred to herein as “Mycoagar.”

The preferred embodiment of the present invention is a combinationpackage containing both CMIT and MCC. Tables 1 and 2 show that forstability of the CMIT and for suppressing growth of mycobacteria, theMCC must be present before the CMIT is added to the MWF or in a mixturewith the CMIT. The results further show that for efficacious biocidalactivity against mycoplasma, the amount of MCC should be greater than250 ppm, preferably about 500 ppm or more, and the amount of CMIT shouldbe greater than 7.5 ppm, preferably 15 ppm or more. Note that the MCCsignificantly enhanced the stability of the CMIT when it was provided ata concentration of about 500 ppm to the MWF before addition of the CMITor in a mixture with the CMIT.

TABLE 1 Stability of CMIT in MWF ppm CMIT Time After Addition Biocideand 0 hr 24 hr 48 hr Concentration MWF 1 MWF 2 MWF 1 MWF 2 MWF 1 MWF 2No biocide <1 <1 <1 <1 <1 <1 7.5 ppm CMIT 8.3 8.4 4.2 4.5 3.2 1.8 15 ppmCMIT 14.8 16.3 5.8 8.5 2.0 2.8 7.5 ppm CMIT/250 ppm Cu 7.7 8.1 7.0 7.75.3 6.5 citrate added together 15 ppm CMIT/500 ppm Cu 13.7 14.0 13.413.5 10.2 12.3 citrate added together 7.5 ppm CMIT added 24 hrs 8.3 4.83.7 3.1 3.3 2.9 before 250 ppm Cu citrate added 15 ppm CMIT added 24 hrs6.6 7.1 6.5 5.8 1.9 4.4 before 500 ppm Cu citrate added 7.5 ppm CMITadded 24 hrs 8.0 7.7 7.9 6.1 6.7 4.9 after 250 ppm Cu citrate added 15ppm CMIT added 24 hrs 14.6 14.5 13.7 13.3 12.3 11.7 after 500 ppm Cucitrate added CMIT: chloromethylisothiazolone (1.5% active ingredient)Stability indicated by bold-faced type

TABLE 2 Anti-Mycobacterial Activity Biocide Mycobacteria/mL and MWF 1MWF 2 Concentration 0 hr 48 hr 0 hr 48 hr No biocide 1.0 × 10⁶ 1.0 × 10⁶3.5 × 10⁵ 3.0 × 10⁶ 7.5 ppm CMIT 1.0 × 10⁶ 1.9 × 10⁵ 3.5 × 10⁵ 1.3 × 10⁵15 ppm CMIT 1.0 × 10⁶ 4.5 × 10³ 3.5 × 10⁵ 4.5 × 10³ 7.5 ppm CMIT/250 ppm1.0 × 10⁶ 1.2 × 10⁴ 3.5 × 10⁵ 8.5 × 10³ Cu citrate added together 15 ppmCMIT/500 ppm 1.0 × 10 ⁶ <100 <100 <100 Cu citrate added together 7.5 ppmCMIT added 24 1.0 × 10⁶ 1.4 × 10⁵ 3.5 × 10⁵ 1.6 × 10⁵ hrs before 250 ppmCu citrate added 15 ppm CMIT added 24 1.0 × 10⁶ 5.0 × 10³ 3.5 × 10⁵ 4.5× 10³ hrs before 500 ppm Cu citrate added 7.5 ppm CMIT added 24 1.0 ×10⁶ 1.2 × 10⁴ 3.5 × 10⁵ 8.0 × 10³ hrs after 250 ppm Cu citrate added 15ppm CMIT added 24 1.0 × 10 ⁶ <100 3.5 × 10 ⁵ <100 hrs after 500 ppm Cucitrate added CMIT: chloromethylisothiazolone (1.5% active ingredient)Effective kill indicated by bold-faced type

EXAMPLES 3 AND 4

In this example, the amount of copper citrate was decreased in a staticsystem to reproduce the potential loss of copper citrate from MWFs in ametalworking plant's recirculating system.

The MWFs selected were fluids which had evidence of CMITincompatibility. The MCC was added to the MWFs 24 hours before the CMITwas added at 15 ppm. Stability was evaluated at time 0 and then after 24and 48 hours by chemical analysis. Plating for mycobacteria kill wasperformed with aliquots from time 0 and after 48 hours.

As shown in Tables 3 and 4, the amount of copper which is needed forstability and effective suppression of mycobacteria growth is greaterthan 250 ppm, preferably about 500 ppm or more.

This example further demonstrates the ability of the 15 ppm CMIT/500 ppmMCC of the present invention to control the Mycobacterium immunogenumisolate which is the etiological agent for HP among machine toolworkers. In addition, the CMIT in the mixture of the present inventionhas increased stability over CMIT mixtures which do not contain the MCC.

TABLE 3 Stability of CMIT in MWF ppm CMIT Time After AdditionConcentration of Cu 0 hr 24 hr 48 hr Citrate in Biocide MWF 1 MWF 2 MWF1 MWF 2 MWF 1 MWF 2 15 ppm CMIT added 24 hrs 13.8 12.9 2.4 2.2 1.8 0.8after 125 ppm Cu citrate added 15 ppm CMIT added 24 hrs 13.7 13.1 2.92.5 1.2 0.8 after 250 ppm Cu citrate added 15 ppm CMIT added 24 hrs 14.614.5 13.7 13.3 12.3 11.7 after 500 ppm Cu citrate added CMIT:chloromethylisothiazolone (1.5% active ingredient) Stability indicatedby bold-faced type

TABLE 4 Anti-Mycobacterial Activity Mycobacteria/mL Concentration of Cu0 hr 24 hr Citrate in Biocide MWF 1 MWF 2 MWF 1 MWF 2 15 ppm CMIT added24 2.0 × 10⁶ 4.5 × 10⁶ 1.0 × 10⁴ 1.0 × 10⁴ hrs before 125 ppm Cu citrateadded 15 ppm CMIT added 24 1.5 × 10⁶ 1.5 × 10⁶ 2.5 × 10³ 1.5 × 10³ hrsafter 250 ppm Cu citrate added 15 ppm CMIT added 24 1.0 × 10 ⁶ 3.5 × 10⁵ <100 <100 hrs after 500 ppm Cu citrate added CMIT:chloromethylisothiazolone (1.5% active ingredient) Effective killindicated by bold-faced type

EXAMPLES 5-8

The data shown in the following tables was generated from samples fromthe field already treated with two biocides, GROTON (a formalydehydecondensate available from Troy Corporation, Florham Park, N.J.) andKATHON (CMIT). Each table shows the results for samples obtained from aparticular source. The tables show the survival of mycobacteria in thepresence of CMIT alone. Note that there are two columns for the CMIT:chlorinated and unchlorinated. The ratio of chlorinated to unchlorinatedin virgin CMIT is 3 to 1. The chlorinated form is the most activecomponent of the CMIT and it is the most nucleophile sensitive. When theratio of chorinated to unchlorinated is less than 3 to 1, it is assumedthat the chlorinated moiety has been reduced by one or more componentsin the MWF.

The tables also show that the total plate count for bacteria for allsamples except for those labeled J were less than 1. The J samples hadno detectable biocides whereas all the other samples had substantiallevels of GROTAN and evidence of CMIT use. GROTAN and CMIT arecompatible biocides.

The data shown in this example demonstrate that mycobacteria are moreresistant to CMIT than other bacteria which can be controlled by lowerlevels of CMIT.

TABLE 5 Total Plate Total Fungal Total Myco Count Count Count SampleBacterial/mL Fungi/mL Myco/mL AFB Stain V1 <1 <10 1.0 × 10³ Very high V2<1 <10 4.0 × 10³ Very high V3 <1 <10 <1 Moderate V4 <1 <10 2.5 × 10²Very high ppm KATHON Sample ppm GROTAN Unchlorinated Chlorinated TotalV1 1837 0 0 0 V2 1402 7.2 15.2 22.4 V3 1119 6.3 15.5 21.8 V4 1837 5.413.8 19.2 AFB: Pellet Stain Counts include live and dead mycobacteria

TABLE 6 Total Plate Total Fungal Total Myco Count Count Count SampleBacterial/mL Fungi/mL Myco/mL AFB Stain V1 <1 <10 7.5 × 10⁵ Moderate V2<1 4.0 × 10¹ 8.5 × 10⁵ Moderate V3 <1 3.0 × 10¹ 1.0 × 10⁴ Low/ModerateV4 <1 <10 2.0 × 10⁴ Very high E <1 1.0 × 10¹ 2.0 × 10⁶ Moderate H₂O <1<10 <1 Negative Solution <1 <10 <1 Negative ppm KATHON Sample ppm GROTANUnchlorinated Chlorinated Total V1 2878 5.3 0.7 6 V2 2518 4.1 0.1 4.2 V33058 1.7 0.2 1.9 V4 2518 5.3 0.5 5.8 E 2698 0 0 0 H₂O   0 0 0 0 Solution2698 0 0 0 AFB: Pellet Stain Counts include live and dead mycobacteria

TABLE 7 Total Plate Total Fungal Total Myco Count Count Count SampleBacterial/mL Fungi/mL Myco/mL AFB Stain V1 <1 <10 2.0 × 10⁵ Very high V2<1 <10 2.5 × 10⁵ Very high E <1 <10 8.0 × 10⁴ Moderate J 3.5 × 10⁴ <10<1 Very high ppm KATHON Sample ppm GROTAN Unchlorinated ChlorinatedTotal V1 2297 11.2 1.9 13.1 V2 2702 8 1.6 9.6 E 3513 7.1 1.4 8.5 J   0 00 0 AFB: Pellet Stain Counts include live and dead mycobacteria

TABLE 8 Total Plate Total Fungal Total Myco Count Count Count SampleBacterial/mL Fungi/mL Myco/mL AFB Stain V1 <1 <10 1.0 × 10⁵ Very high V2<1 <10 4.0 × 10⁵ Very high V3 <1 <10 2.0 × 10⁵ Moderate/high V4 <1 <101.0 × 10⁵ Very high E <1 <10 2.5 × 10⁵ Moderate/high J 1.5 × 10⁷ 1.1 ×10² <1 Moderate ppm KATHON Sample ppm GROTAN Unchlorinated ChlorinatedTotal V1 1417 9.5 1.8 11.3 V2 2083 7.1 1.3 8.4 V3 2000 0 0 0 V4 2417 81.3 9.3 E 2000 7.5 1 8.5 J   0 0 0 0 AFB: Pellet Stain Counts includelive and dead mycobacteria

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the claims attached herein.

1. A method for suppressing growth of mycobacteria includingMycobacterium immunogenum associated with hypersensitivity pneumonitisin an industrial metalworking fluid which comprises: (a) providing acomposition comprising a copper complex comprising disodium monocopper(II) citrate and an isothiazolone mixture comprising5-chloro-2-methyl-4-isothiazolin-3-one and2-methyl-4-isothiazolin-3-one, wherein the copper complex providescopper ion to the used metalworking fluid in an amount which maintainsbiocidal activity of the isothiazolone mixture; (b) adding the coppercomplex and isothiazolone to the metalworking fluid which containscomponents from use of the fluid which interfere with the antimicrobialactivity of isothiazoline mixture in an amount which is sufficient tosuppress the growth of the mycobacteria in the metalworking fluid for atleast 48 hours; and (c) monitoring the metalworking fluid containing thecomposition for the mycobacteria to determine that the mycobacteria havebeen suppressed.
 2. The method of claim 1 wherein the isothiazolonemixture is present in an amount which is less than is required for thebiocidal activity when the isothiazolone mixture is introduced into themetalworking fluid alone.
 3. The method of claim 1 wherein the5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-oneare in a weight ratio of about 3 to
 1. 4. The method of claim 1 whereinthe composition can be mixed with the metalworking fluid to produce anamount of the isothiazolone mixture which is from about 15 ppm to about21 ppm.
 5. The method of claim 1 wherein the Mycobacterium immunogenumhas a restriction fragment length polymorphism pattern produced by aDraI digest of Mycobacterium immunogenum DNA as shown in FIG.
 1. 6. Themethod of claim 1 wherein the copper complex provides about 500 ppmcopper ion to the metalworking fluid.
 7. The method of claim 1 whereinthe isothiazolone is incompatible with the metalworking fluid when theisothiazolone mixture is introduced into the metalworking fluid alone.8. A method for suppressing growth of mycobacteria includingMycobacterium immunogenum in a metalworking fluid which comprises: (a)providing a copper complex comprising disodium monocopper (II) citrateand an isothiazolone mixture comprising5-chloro-2-methyl-4-isothiazolin-3-one 2-methyl-4-isothiazolin-3-one,wherein the copper complex provides more than about 250 ppm of copperion to the used metalworking fluid; (b) adding the copper complex andthe isothiazolone mixture to the metalworking fluid which containscomponents from use of the fluid which interfere with the antimicrobialactivity of isothiazolone mixture, wherein the metal complex and theisothiazolone mixture suppresses the growth of the mycobacteria in theused metalworking fluid for at least 48 hours; and (c) monitoring themetalworking fluid containing the metal complex and isothiazolone forthe mycobacteria to determine that the mycobacteria have beensuppressed.
 9. The method of claim 8 wherein the isothiazolone mixtureis present in an amount which is less than is required for biocidalactivity when the isothiazolone mixture is introduced into themetalworking fluid alone.
 10. The method of claim 8 wherein the coppercomplex is added to the metalworking fluid up to about 24 hours beforethe isothiazolone mixture is added to the metalworking fluid.
 11. Themethod of claim 8 wherein the copper complex and the isothiazolonemixture are added to the metalworking fluid at the same time.
 12. Themethod of claim 8 wherein the 5-chloro-2-methyl-4-isothiazolin-3-one and2-methyl-4-isothiazolin-3-one are in a weight ratio of about 3 to
 1. 13.The method of claim 8 wherein the composition can be mixed with themetalworking fluid to produce an amount of the isothiazolone mixturewhich is from about 15 ppm to about 21 ppm.
 14. The method of claim 8wherein the Mycobacterium immunogenum has a restriction fragment lengthpolymorphism pattern produced by a DraI digest of Mycobacteriumimmunogenum DNA as shown in FIG.
 1. 15. The method of claim 8 whereinthe isothiazolone is incompatible with the metalworking fluid when theisothiazolone mixture is introduced into the metalworking fluid alone.